Amplicon generation

Abstract

The invention provides compositions and methods for accurately and specifically amplifying sequences to allow for accurate and specific detection of mutations, such as in disease-associated genes and alleles, thus distinguishing true nucleotide variants over random nucleotide sequencing errors. In one embodiment, this is accomplished, prior to sequencing, by using a combination of director and driver in a combination of two PCR reactions. Thus, in one embodiment, this is accomplished by amplifying a nucleotide sequence of interest to introduce a director that creates a specific target for a subsequent amplification in which a driver that specifically hybridizes to the director drives the specificity of further amplification. The amplification produces an amplicon of a sense or antisense strand of a double-stranded nucleotide sequence of interest. The amplicon may optionally contain universal sequences and / or index sequences that facilitate subsequent sequencing of the amplicon, such as using sequencing-by-synthesis. The reagents of the first and second amplification steps may be combined in a single reaction mixture.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority under 35 U.S.C. § 119(e) to co-pending U.S. Provisional Patent Application Ser. No. 62 / 798,163, filed Jan. 29, 2019, incorporated by reference.SEQUENCE LISTING[0002]A Sequence Listing has been submitted in an ASCII text file named “19594” created on May 10, 2020, consisting of 34,802 bytes, the entire content of which is herein incorporated by reference.FIELD OF THE INVENTION[0003]The invention provides compositions and methods for accurately and specifically amplifying sequences to allow for accurate and specific detection of mutations, such as in disease-associated genes and alleles, thus distinguishing true nucleotide variants over random nucleotide sequencing errors. In one embodiment, this is accomplished, prior to sequencing, by using a combination of director and driver in a combination of two PCR reactions. Thus, in one embodiment, this is accomplished by first amplifying a nucleotide sequence of i...

AI Technical Summary

Benefits of technology

The invention provides methods for amplifying and sequencing specific parts of DNA associated with disease, such as the HLA DQA and DQB genes associated with celiac disease. These methods use a combination of a driver and a director to efficiently produce sequenceable amplicons, which can be easily detected and sequenced. The methods also accurately detect the specific variants associated with disease, reducing the need for amplifying and sequencing the entire gene. Additionally, the methods can be performed using a single reaction vessel, reducing the likelihood of transcription errors and secondary PCR contamination. The methods also generate amplicons that are complementary to the flowcell surface, allowing for more accurate sequencing. Overall, the methods are efficient, accurate, and cost-effective for detecting disease-associated variants.

Problems solved by technology

Testing for celiac disease is one of the highest volume assays, costly, and time consuming, requiring a PCR, a gel, hybridization and washing of beads, and reading on instruments.

Thus, even small cost savings in this assay would lead to large overall savings.

Current genetic testing for celiac disease can rule out celiac disease, can indicate an individual is at risk to develop celiac disease, but cannot, alone, directly diagnose celiac disease.

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